Highly active mutant enzyme for producing 4-hydroxybenzoic acid or salt thereof

ABSTRACT

The mutant chorismate-pyruvate lyase (A) or (B) as described below is capable of producing 4-hydroxybenzoic acid or a salt thereof with sufficient practical efficiency. 
     (A) A mutant chorismate-pyruvate lyase obtained by replacing the valine at position 80 in a chorismate-pyruvate lyase (ubiC) from  Pantoea ananatis  consisting of the amino acid sequence of SEQ ID NO: 1 with one or more other amino acids. 
     (B) A mutant chorismate-pyruvate lyase obtained by replacing an amino acid in another chorismate-pyruvate lyase, the amino acid being at a position enzymologically homologous with that of the above valine, with one or mere other amino acids.

TECHNICAL FIELD

The present invention provides a mutant enzyme modified for improvedproductivity to produce 4-hydroxybenzoic acid or a salt thereof(hereinafter may be abbreviated as “4-HBA”), a coryneform bacteriumtransformant having an improved 4-HBA productivity as a result of higherexpression of the mutant enzyme, and an efficient 4-HBA-producingprocess using the transformant.

BACKGROUND ART

Against the backdrop of global warming and exhaustion of fossilresources, production of chemical products using renewable resources hasattracted attention as an emerging industry for realizing a low-carbonsociety.

4-HBA is a useful substance used as a raw material for liquid-crystalpolymers, as a raw material for the synthesis of paraben, which is anantimicrobial agent, and the like.

Currently, 4-HBA is produced by chemical conversion from crude oil as araw material. Examples of chemical 4-HBA production processes include aprocess in which phenol, potassium hydroxide, and carbon dioxide arereacted under high-pressure conditions.

Such a process depends on fossil materials for phenol as the startingmaterial, and in addition, places a great burden on the environmentbecause the process requires strong alkali, carbon dioxide, andhigh-temperature and high-pressure conditions, and produces hazardousliquid waste.

Therefore, there is a strong need to establish an energy-saving,environment-conscious process that allows biological production of 4-HBAand produces a reduced amount of hazardous liquid waste.

However, biological production of 4-HBA from renewable resources is lessproductive as compared to production of lactic acid or ethanol becausethe metabolic reaction from a raw material sugar consists of a greatmany steps. In addition, there are problems, such as inhibition ofbacterial growth by produced 4-HBA and cytotoxicity of 4-HBA. Therefore,industrial production of 4-HBA has not been achieved.

Using Escherichia coli, it has been revealed that 4-HBA is synthesizedfrom chorismic acid, which is an intermediate in the shikimate pathwayinvolved in the synthesis of aromatic amino acids etc., bychorismate-pyruvate lyase encoded by ubiC (Non Patent Literature 1 and2, Patent Literature 1 and 2).

There is a report of introduction of a chorismate pyruvate-lyase gene(ubiC) of Escherichia coli into a different kind of microorganism,Klebsiella pneumoniae, as a host, in an attempt to produce 4-HBA (NonPatent Literature 3). Also, there is a report of fermentative productionof 4-HBA in an Escherichia coli in which the shikimic acid pathway isreinforced (Non Patent Literature 4). In an attempt to avoid the growthinhibition or the toxic action by 4-HBA, there are reports of selectionof 4-HBA-resistant strains and of culture in the presence of anion-exchange resin, but practically sufficient 4-HBA productivity hasnot been achieved (Non Patent Literature 2).

Regarding ubiCs of other living organisms than Escherichia coli, theubiC of Rhodobacter sphaeroides has been reported. However, anEscherichia coli transformant highly expressing ubiC and a Rhodobactersphaeroides transformant highly expressing ubiC are capable of producing4-HBA only at low concentrations, which are not practically sufficient(Patent Literature 3). Also, despite the description that the ubiC ofRhodobacter sphaeroides can complement the ubiC of Escherichia coli in adisruptant of Escherichia coli lacking the ubiC gene, the literaturedoes not include any enzymatic activity values, description regardingenzymatic characteristics, or detailed description regarding comparisonwith enzymes from other living organisms.

The UbiC of Escherichia coli has already been enzymatically analyzed indetail, and is known to be strongly inhibited by the product, 4-HBA(product inhibition) (Non Patent Literature 2 and 5). Therefore, inorder to establish a high 4-HBA-producing strain aiming at a higherproduction of 4-HBA, obtaining a highly active ubiC and obtaining aresistant ubiC against product inhibition by 4-HBA are extremelyimportant.

CITATION LIST Non Patent Literature

Non Patent literature 1: J. Bacteriol., 174, 5309-5316 (1992)

Non Patent literature 2: Microbiology, 140, 897-904 (1994)

Non Patent literature 3: Appl. Microbiol. Biotechnol., 43, 985-988(1995)

Non Patent literature 4: Biotechnol. Bioeng., 76, 376-390 (2001)

Non Patent Literature 5: Biochimica et Biophysica Acta, 1594, 160-167(2002)

Patent Literature

Patent literature 1: U.S. Pat. No. 6,030,819

Patent literature 2: U.S. Pat. No. 6,114,157

Patent literature 3: JP 2012-183048 A

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a mutantchorismate-pyruvate lyase having a practically sufficient level of4-KBA-producing activity; a transformant which highly expresses theenzyme and thereby is capable of efficiently producing 4-HBA; and aprocess for efficiently producing 4-HBA using the transformant.

Solution to Problem

The present inventors have wholeheartedly carried out investigations inorder to achieve the object described above and found that a mutantchorismate-pyruvate lyase having an. improved ability to produce 4-HBAfrom glucose, chorismic acid, or the like can be obtained by introducinga mutation to a ubiC gene, which mutation being replacement of

(A) valine at position 80 in a chorismate-pyruvate lyase (ubiC) fromPantoea ananatis consisting of the amino acid sequence of SEQ ID NO: 1,or

(B) an amino acid in another chorismate-pyruvate lyase, the amino acidbeing at a position enzymologically homologous with that of the abovevaline, with a different amino acid.

The present invention, which has been completed based on theabove-mentioned findings, provides the following mutantchorismate-pyruvate lyases, transformants and processes for producing4-HBA.

[1] A mutant chorismate-pyruvate lyase of the following (A) or (B).

(A) A mutant chorismate-pyruvate lyase obtained by replacing valine atposition 80 in a chorismate-pyruvate lyase (ubiC) from Pantoea ananatisconsisting of the amino acid sequence of SEQ ID NO: 1 with one or moreother amino acids.

(B) A mutant chorismate-pyruvate lyase obtained by replacing an aminoacid in another chorismate-pyruvate lyase, the amino acid being at aposition enzymologically homologous with that of the above valine, withone or more other amino acids.

[2] The mutant chorismate-pyruvate lyase of the above [1], wherein themutant chorismate-pyruvate lyase (B) is obtained by replacing

(i) valine (V) at position 80 in a chorismate-pyruvate lyase from aProvidencia bacterium,

(ii) isoleucine (I) at position 79 in a chorismate-pyruvate lyase froman Escherichia bacterium, or

(iii) isoleucine (I) at position 79 in a chorismate-pyruvate lyase froma Cronobacter bacterium with one or more other amino acids.

[3] The mutant chorismate-pyruvate lyase of the above [1], which is thefollowing (a), (b), or (c):

(a) a mutant chorismate-pyruvate lyase obtained by replacing valine atposition 80 in a chorismate-pyruvate lyase from Pantoea ananatisconsisting of the amino acid sequence of SEQ ID NO: 1, valine atposition 80 in a chorismate-pyruvate lyase from Providencia stuartiiconsisting of the amino acid sequence of SEQ ID NO: 2, valine atposition 80 in a chorismate-pyruvate lyase from Providencia rustigianiiconsisting of the amino acid sequence of SEQ ID NO: 3, valine atposition 80 in a chorismate-pyruvate lyase from Providencia sneebiaconsisting of the amino acid sequence of SEQ ID NO: 4, valine atposition 80 in a chorismate-pyruvate lyase from Providencia rettgericonsisting of the amino acid sequence of SEQ ID NO: 5, valine atposition 80 in a chorismate-pyruvate lyase from Providenciaalcalifaciens consisting of the amino acid sequence of SEQ ID NO: 6, orvaline at position 80 in a chorismate-pyruvate lyase from Providenciaburhodogranariea consisting of the amino acid sequence of SEQ ID NO: 7,

isoleucine at position 79 in a chorismate-pyruvate lyase fromEscherichia coli consisting of the amino acid sequence of SEQ ID NO: 8,or

isoleucine at position 79 in a chorismate-pyruvate lyase fromCronobacter sakazakii consisting of the amino acid sequence of SEQ IDNO: 9

with one or more other amino acids;

(b) a mutant chorismate-pyruvate lyase, which consists of an amino acidsequence having the amino-acid segment introduced by the abovereplacement and having 90% or more of identity with any one of SEQ IDNOs: 1 to 9, and has chorismate-pyruvate lyase activity; or

(c) a mutant chorismate-pyruvate lyase as a polypeptide which consistsof an amino acid sequence having 90% or more of identity with any one ofSEQ ID NOs: 1 to 9, has chorismate-pyruvate lyase activity, and has areplacement of an amino acid at a position enzymologically homologouswith that of valine at position 80 of any one of SEQ ID NOs: 1 to 7 orisoleucine at position 79 of SEQ ID NO: 8 or 9 with one or more otheramino acids.

[4] The mutant chorismate-pyruvate lyase of any one of the above [1] to[3], wherein the valine or isoleucine is replaced with one amino acidwhich is alanine, cysteine, threonine, serine, or asparagine.[5] A transformant obtained by introducing, into a coryneform bacteriumas a host, a DMA which encodes the mutant chorismate-pyruvate lyase ofany one of the above [1] to [4].[6] The transformant of the above [5], wherein the coryneform bacteriumas the host is a Corynebacterium.[7] The transformant of the above [6], wherein the Corynebacteriumglutamicum is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032,crATCC13869.[8] A transformant obtained by introducing the following mutant DMA:

(C) a mutant DNA obtained by replacing gtc at positions 240 to 242 in achorismate-pyruvate lyase (ubiC) gene of Pantoea ananatis consisting ofthe base sequence of SEQ ID NO: 10 with a DNA segment which encodes oneor more amino acids different from the amino acid encoded by gtc; or

(D) a mutant DNA obtained by replacing a DNA segment in a gene whichencodes another chorismate-pyruvate lyase, the DNA segment being atpositions corresponding to the above gtc, with a DNA segment whichencodes one or more amino acids different from the amino acid encoded bythe original DNA segment into a coryneform bacterium as a host.

[9] The transformant of the above [8]wherein the DNA of the above (D) isa mutant DNA obtained by replacing

(iv) gtc at positions 240 to 242 in a chorismate-pyruvate lyase gene ofa Providencia bacterium,

(v) ate at positions 237 to 239 in a chorismate-pyruvate lyase gene ofan Escherichia bacterium, or

(vi) ate at positions 237 to 239 in a chorismate-pyruvate lyase gene ofa Cronobacter bacterium

with a DNA segment which encodes one or more amino acids different fromthe amino acid encoded by the original DNA segment.[10] The transformant of the above [8]wherein the mutant DNA is thefollowing (d), (e), or (f).

(d) A mutant DNA obtained by replacing gtc at positions 240 to 242 in achorismate-pyruvate lyase gene of Pantoea ananatis consisting of thebase sequence of SEQ ID NO: 10, a chorismate-pyruvate lyase gene ofProvidencia stuartii consisting of the base sequence of SEQ ID MO: 11, achorismate-pyruvate lyase gene of Providencia rustigianii consisting ofthe base sequence of SEQ ID NO: 12, a chorismate-pyruvate lyase gene ofProvidencia sneebia consisting of the base sequence of SEQ ID NO: 13, achorismate-pyruvate lyase gene of Providencia rettgeri consisting of thebase sequence of SEQ ID NO: 14, a chorismate-pyruvate lyase gene ofProvidencia alcalifaciens consisting of the base sequence of SEQ ID NO:15, or a chorismate-pyruvate lyase gene of Providencia burhodogranarieaconsisting of the base sequence, of SEQ ID NO: 16 with a DNA segmentwhich encodes one or more amino acids different from valine,

a mutant DNA obtained by replacing ate at positions 237 to 239 in achorismate-pyruvate lyase gene of Escherichia coli consisting of thebase sequence of SEQ ID NO: 17 with a DNA segment which encodes one ormore amino acids different from isoleucine, or

a mutant DNA obtained by replacing ate at positions 237 to 239 in achorismate-pyruvate lyase gene of Cronobacter sakazakii consisting ofthe base sequence of SEQ ID NO: 18 with a DNA segment which encodes oneor more amino acids different from isoleucine.

(e) A mutant DNA, which consists of a base sequence having the DNAsegment introduced by the above replacement and having 90% or more ofidentity with any one of SEQ ID NOs: 10 to 18, and encodes a polypeptidehaving chorismate-pyruvate lyase activity.

(f) A mutant DNA which consists of a base sequence having 90% or more ofidentity with any one of SEQ ID NOs: 10 to 18; encodes a polypeptidehaving chorismate-pyruvate lyase activity; and has a replacement of gtcat positions 240 to 242 in any one of SEQ ID NOs: 10 to 16 with a DNAsegment which encodes one or more amino acids different from valine, ora replacement of a DNA segment; corresponding to ate at positions 237 to239 in SEQ ID NO: 17 or 18 with a DNA segment which encodes one or moreamino acids different from isoleucine

(here, the DNA segment at positions 240 to 242 in any one of SEQ ID NOs:10 to 16 corresponding to gtc is a DNA which encodes an amino acid at aposition enzymologically homologous with that of valine at position 80of a chorismate-pyruvate lyase encoded by a DNA consisting of the basesequence of any one of SEQ ID NOs: 10 to 16; and the DNA segmentcorresponding to gtc at positions 237 to 239 in SEQ ID NO: 17 or 18 is aDNA which encodes an amino acid at a position enzymologically homologouswith that of isoleucine at position 79 of a chorismate-pyruvate lyaseencoded by a DNA consisting of the base sequence of SEQ ID NO: 17 or18).[11] The transformant of any one of the above [8] to [10], wherein theDNA introduced by the above replacement is gca, tgc, acc, tcc, or aac.[12] The transformant of any one of the above [8] to [11], wherein thecoryneform bacterium as the host is a Corynebacterium.[13] The transformant of the above [12], wherein the Corynebacteriumglutamicum is Corynebacterium glutamicum R (FERM BP-18976), ATCC13032,crATCC13S69.[14] Corynebacterium glutamicum HBA-47 (Accession Number: NITEBP-01849), which is a transformant of Corynebacterium glutamicum.[15] A process for producing 4-hydroxybenzoic acid or a salt thereof,which comprises a step of culturing the transformant of any one of theabove [5] to [14] in a reaction mixture containing at least one startingcompound selected from the group consisting of a sugar, a compound thatcan be metabolized into chorismic acid by the transformant, chorismicacid, and a salt thereof, and a step of recovering 4-hydroxybenzoic acidor a salt thereof from the reaction mixture.[16] The process of the above [15], wherein the transformant is culturedunder aerobic conditions where the transformant does not grow.

Advantageous Effects of Invention

Using the mutant chorismate-pyruvate lyase of the present invention, ora coryneform bacterium transformant obtained by introducing a DNA whichencodes the mutant chorismate-pyruvate lyase gene, 4-HBA can be producedwith sufficient practical efficiency from a sugar, a compound that canbe metabolized into chorismic acid by the transformant, chorismic acid,and a salt thereof.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 shows the effect of 4-hydroxybenzoic acid on the growth of fourkinds of microorganisms (Corynebacterium glutamicum R, Escherichia coliJM109, Pseudomonas putida S12 ATCC7008G1, and Rhodobacter sphaeroidesNBRC12203).

DESCRIPTION OF EMBODIMENTS

Hereinafter, the present invention will be described in detail.

(1) Mutant Chorismate-Pyruvate Lyase

The mutant chorismate-pyruvate lyase of the present invention is

(A) a mutant chorismate-pyruvate lyase obtained by replacing valine atposition 80 in a chorismate-pyruvate lyase (ubiC) from Pantoea ananatisconsisting of the amino acid sequence of SEQ ID NO: 1 with another aminoacid(s), or

(B) a mutant chorismate-pyruvate lyase obtained by replacing an aminoacid in another chorismate-pyruvate lyase, the amino acid being at aposition enzymologically homologous with that of the above valine, withanother amine acid(s).

Examples of the above mutant chorismate-pyruvate lyase (B) include amutant chorismate-pyruvate lyase obtained by replacing

(i) valine (V) at position 80 in a chorismate-pyruvate lyase from aProvidencia bacterium,

(ii) isoleucine (I) at position 79 in a chorismate-pyruvate lyase froman Escherichia bacterium, or

(iii) isoleucine (I) at position 79 in a chorismate-pyruvate lyase froma Cronobacter bacterium with another amino acid(s).

Examples of the chorismate-pyruvate lyase from a Providencia bacteriuminclude a chorismate-pyruvate lyase from Providencia stuartii consistingof the amino acid sequence of SEQ ID NO: 2, a chorismate-pyruvate lyasefrom Providencia rustigianii consisting of the amino acid sequence ofSEQ ID NO: 3, a chorismate-pyruvate lyase from Providencia sneebiaconsisting of the amino acid sequence of SEQ ID NO: 4, achorismate-pyruvate lyase from Providencia rettgeri consisting of theamino acid sequence of SEQ ID NO: 5, a chorismate-pyruvate lyase fromProvidencia alcalifaciens consisting of the amino acid sequence of SEQID NO: 6, and a chorismate-pyruvate lyase from Providenciaburhodogranariea consisting of the amino acid sequence of SEQ ID NO: 7.

Examples of the chorismate-pyruvate lyase from an Escherichia bacteriuminclude a chorismate-pyruvate lyase from Escherichia coli consisting ofthe amino acid sequence of SEQ ID NO: 8.

Examples of the chorismate-pyruvate lyase from a Cronobacter bacteriuminclude a chorismate-pyruvate lyase from Cronobacter sakazakiiconsisting of the amino acid sequence of SEQ ID NO: 9.

The above-mentioned valine or isoleucine may be replaced with anotheramino acid or two or more (for example, 2 to 8, 2 to 6, 2 to 3, or 2)amino acids. Preferably, the valine or isoleucine is replaced with oneamino acid. In particular, the valine or isoleucine is preferablyreplaced with alanine (A), cysteine (C), threonine (T), serine (S), orasparagine (N), and more preferably replaced with alanine (A) orcysteine (C).

Instead of the replacement of the valine or isoleucine, deletion of thevaline or isoleucine, deletion of one or more (for example, 2 :o 8, 2 to6, 2 to 3, or 2) amino acids adjacent to the N-terminal side of thevaline or isoleucine, or insertion of one or more (for example, 2 to 8,2 to 6, 2 to 3, or 2) amino acids to the position adjacent to the valineor isoleucine can also be employed to enhance the chorismate-pyruvatelyase activity.

The mutant chorismate-pyruvate lyase of the present invention includes amutant chorismate-pyruvate lyase which consists of a polypeptide havingthe amino-acid segment introduced by the mutation (for example, alanine,cysteine, threonine, serine, or asparagine) and having 90% or more,preferably 95% or more, more preferably 98% or more of identity with anyone of SEQ ID NOs: 1 to 9, and has chorismate-pyruvate lyase activity.That is, in the mutant chorismate-pyruvate lyase, the amino acid at aposition enzymologically homologous with that of the valine at position80 of any one of SEQ ID NOs: 1 to 7 or the isoleucine at position 75 ofSEQ ID NO: 8 or 9 is not valine or isoleucine but alanine, cysteine,threonine, serine, or asparagine, for example.

In the present invention, “enzymologically homologous” means beingequivalent in contribution to the enzymatic function of the polypeptide.The amino acid located at an equivalent position in an amino-acidalignment will equivalently contribute to the enzymatic function.

In the present invention, the homologies of amino acid sequences and thehomologies of base sequences were calculated using GENETYX Ver. 8 (madeby Genetyx).

Chorismate-pyruvate lyase is an enzyme that catalyzes a reaction inwhich 4-HBA is produced by elimination of pyruvic acid from chorismicacid and the reverse reaction thereof.

The chorismate-pyruvate lyase activity can be measured by an alteredmethod of the method described in “Microbiology, 140, 897-904 (1994)”.Briefly, the enzyme to be tested is added to a test solution containing50 mM of Tris-HCl (pH 7.5), 0.5 mM of chorismate Ba salt, 0.2 mM ofNADH, 0.2 M of NaCl and 5 units of lactate dehydrogenase, the reactionis allowed to proceed at 33° C., and the decrease in absorbance of NADHat 340 nm is monitored to determine the initial rate of the reaction.Using a system not containing the chorismate Ba salt, the reaction isperformed in a similar manner to obtain background values. Thedifference between the measurements is considered to result from thechorismate-pyruvate lyase activity. When linear reduction in theabsorbance of NADH at 340 nm with time is observed (which reductiondepends on the enzyme and the substrate added), chorismate-pyruvatelyase activity is judged to exist. One unit of enzyme activity isdefined as the amount of the enzyme that produces 1 μmol of 4-HBA perminute, and is calculated from the initial rate of the enzyme reaction.

The mutant chorismate-pyruvate lyase of the present invention includes amutant chorismate-pyruvate lyase as a polypeptide which has 90% or more,preferably 95% or more, more preferably 98% or more of identity with anyone of SEQ ID NOs: 1 to 9, has chorismate-pyruvate lyase activity, andhas a replacement of an amino acid at a position enzymologicallyhomologous with that of valine at position 30 of any one of SEQ ID NOs:1 to 7 or isoleucine at position 79 of SEQ ID NO: 8 or 9 with one ormore other amino acids (for example, alanine, cysteine, threonine,serine, or asparagine; in particular, alanine or cysteine).

(2) Corynebacterium transformant

By introducing a mutant DNA which encodes the above-described mutantchorismate-pyruvate lyase into a coryneform bacterium as a host, atransformant capable of efficiently producing 4-HBA can be obtained.

Examples of the mutant DNA include a mutant DNA obtained by replacing

(C) gtc at positions 240 to 242 in a chorismate-pyruvate lyase (ubiC)gene of Pantoea ananatis consisting of the base sequence of SEQ ID NO:10; or

(D) a DNA segment at positions corresponding to the above gtc in a genewhich encodes another chorismate-pyruvate lyase with a DNA segment whichencodes one or more (for example, 2 to 8, 2 to 6, 2 to 3, or 2) aminoacids different from the amino acid encoded by the original DNA segment.

Examples of the mutant DNA of the above (D) include a mutant DNAobtained by replacing

(iv) gtc at positions 240 to 242 in a chorismate-pyruvate lyase gene ofa Providencia bacterium,

(v) ate at positions 237 to 239 in a chorismate-pyruvate lyase gene ofan Escherichia bacterium, or

(vi) ate at positions 237 to 239 in a chorismate-pyruvate lyase gene ofa Cronobacter bacterium

with a DNA segment which encodes one or more (for example, 2 to 8, 2 to6, 2 to 3, or 2) different amino acids.

Examples of the chorismate-pyruvate lyase gene of a Providenciabacterium include a chorismate-pyruvate lyase gene of Providenciastuartii consisting of the base sequence of SEQ ID NO: 11, achorismate-pyruvate lyase gene of Providencia rustigianii consisting ofthe base sequence of SEQ ID NO: 12, a chorismate-pyruvate lyase gene ofProvidencia sneebia consisting of the base sequence of SEQ ID NO: 13, achorismate-pyruvate lyase gene of Providencia rettgeri consisting of thebase sequence of SEQ ID NO: 14, a chorismate-pyruvate lyase gene ofProvidencia alcalifaciens consisting of the base sequence of SEQ ID NO:15, and a chorismate-pyruvate lyase gene of Providencia burhodogranarieaconsisting of the base sequence of SEQ ID NO: 16.

Examples of the chorismate-pyruvate lyase gene of an Escherichiabacterium include a chorismate-pyruvate lyase gene of Escherichia coliconsisting of the base sequence of SEQ ID NO: 17.

Examples of the chorismate-pyruvate lyase gene of a Cronobacterbacterium include a chorismate-pyruvate lyase gene of Cronobactersakazakii consisting of the base sequence of SEQ ID NO: 18.

It is particularly preferable that the DNA segment is replaced with aDNA which encodes one amino acid. In particular, the DNA segment ispreferably replaced with a DNA which encodes alanine, cysteine,threonine, serine, or asparagine, and more preferably replaced with aDNA which encodes alanine or cysteine.

Examples of the DNA which encodes alanine include gca, examples of theDNA which encodes cysteine include tgc, examples of the DNA whichencodes threonine include acc, examples of the DNA which encodes serineinclude tcc, and examples of the DNA which encodes cysteine include aac.

The mutant DNA also includes a mutant DNA which consists of a basesequence having the DNA segment introduced by the replacement whichsegment encodes an amino acid that is not valine or isoleucine (forexample, alanine, cysteine, threonine, serine, or asparagine; inparticular, alanine or cysteine) and having 90% or more, preferably 95%or more, more preferably 98% or more of identity with any one of SEQ IDNOs: 10 to 18, and encodes a polypeptide having chorismate-pyruvatelyase activity.

The mutant DNA also includes a mutant DNA which has the DNA segmentintroduced by the replacement which segment encodes an amino acid thatis not valine or isoleucine (for example, alanine, cysteine, threonine,serine, or asparagine; in particular, alanine or cysteine), hybridizesto a DNA consisting of any one of SEQ ID NOs: 10 to 18 under stringent,conditions, and encodes a polypeptide having chorismate-pyruvate lyaseactivity.

In the present invention, “stringent conditions” means conditions inwhich hybridization is performed in a hybridization solution at a saltconcentration of 6× SBC at 50 to 60° C. for 16 hours and then washingwith a solution at 0.1× SSC is performed.

The mutant DNA also includes a mutant DNA which consists of a basesequence having 90% or more, preferably 95% or more, more preferably 98%or more of identity with any one of SEQ ID NOs: 10 to 16; encodes apolypeptide having chorismate-pyruvate lyase activity; and has areplacement of a DNA segment corresponding to gtc at positions 240 to242 in any one of SEQ ID NOs: 10 to 16 with a DNA segment which encodesone or more (for example, 2 to 8, 2 to 6, 2 to 3, or 2) amino acids, inparticular, one amino acid different from valine (for example, alanine,cysteine, threonine, serine, or asparagine; in particular, alanine orcysteine).

“The DNA segment corresponding to gtc at positions 240 to 242 in any oneof SEQ ID NOs: 10 to 16” means a DNA segment which encodes an amino acidat a position enzymologically homologous with that of valine at position80 of chorismate-pyruvate lyase consisting of an amino acid sequenceencoded by a DNA consisting of the base sequence of any one of SEQ IDNOs: 10 to 16.

The mutant DNA also includes a mutant DNA which consists of a basesequence having 90% or more, preferably 95% or more, more preferably 98%or more of identity with SEQ ID NO: 17 or 18; encodes a polypeptidehaving chorismate-pyruvate lyase activity; and has a replacement of aDNA segment corresponding to ate at positions 237 to 239 in SEQ ID NO:17 or 18 with a DNA segment which encodes one or more (for example, 2 to8, 2 to 6, 2 to 3, or 2) amino acids, in particular, one amino aciddifferent from isoleucine (for example, alanine, cysteine, threonine,serine, or asparagine; in particular, alanine or cysteine).

“The DNA segment corresponding to ate at positions 237 to 239 in SEQ IDNO: 17 or 18” means a DNA segment which encodes an amino acid at aposition enzymologically homologous with that of the isoleucine atposition 75 of chorismate-pyruvate lyase encoded by a DNA consisting ofthe base sequence of SEQ ID NO: 17 or 18).

The mutant DNA also includes a mutant DNA which hybridizes to any one ofSEQ ID NOs: 10 to 16 under stringent conditions; encodes a polypeptidehaving chorismate-pyruvate lyase activity; and has a replacement of aDNA segment corresponding to gtc at positions 240 to 242 in any one ofSEQ ID NOs: 10 to 16 with a DNA segment which encodes one or more (forexample, 2 to 8, 2 to 6, 2 to 3, or 2) amino acids, in particular, oneamino acid different from valine (for example, alanine, cysteine,threonine, serine, or asparagine; in particular, alanine or cysteine).

The mutant DNA also includes a mutant. DNA which hybridizes to SEQ IDNO: 17 or 18 under stringent conditions; encodes a polypeptide havingchorismate-pyruvate lyase activity; and has a replacement of a DNAsegment corresponding to ate at positions 237 to 239 in SEQ ID NO: 17 orIS with a DNA segment which encodes one or more (for example, 2 to 8, 2to 6, 2 to 3, or 2) amino acids, in particular, one amino acid differentfrom isoleucine (for example, alanine, cysteine, threonine, serine, orasparagine; in particular, alanine or cysteine).

A DNA analog which has 90% or more of identity with a DNA that encodeschorismate-pyruvate lyase or hybridizes thereto under stringentconditions, and encodes a polypeptide having chorismate-pyruvate lyaseactivity can be selected from, for example, a DNA library of a differentspecies by PCR or hybridization using a primer or a probe designed basedon the base sequence of the original DNA, according to a conventionalmethod, and as a result, a DNA which encodes a polypeptide havingchorismate-pyruvate lyase activity can be obtained with a highprobability.

The coryneform bacteria are a group of microorganisms defined inBergey's Manual of Determinative Bacteriology, Vol. 8, 599 (1974), andare not particularly limited as long as they grow under normal aerobicconditions.

The specific examples include the genus Corynebacterium, the genusBrevibacterium, the genus Arthrobacter, the genus Mycobacterium and thegenus Micrococcus. Among the coryneform bacteria, the genusCorynebacterium is preferred.

Examples of the genus Corynebacterium include Corynebacteriumglutamicum, Corynebacterium efficiens, Corynebacterium ammoniagenes,Corynebacterium halotolerance, and Corynebacterium alkanolyticum. Amongthem, Corynebacterium glutamicum is preferred for safety and high 4-HBAproduction. Examples of preferred strains include Corynebacteriumglutamicum R (FERM BP-18976), ATCC13032, ATCC13869, ATCC13058,ATCC13059, ATCC13060, ATCC13232, ATCC13286, ATCC13287, ATCC13655,ATCC13745, ATCC13746, ATCC13761, ATCC14020, ATCC31831, MJ-233 (FERMBP-1497), and MJ-233AB-41 (FERM BP-1498). These strains are depositedinternationally under the Budapest Treaty and available to the public.

Among them, strains R (FERM BP-18976), ATCC13032, and ATCC13869 arepreferred.

According to molecular biological classification, names of some speciesof coryneform bacteria, such as Brevibacterium flavum, Brevibacteriumlactofermentum, Brevibacterium divaricatum, and Corynebacterium liliumare standardized to Corynebacterium glutamicum (Liebl, W, et al.,Transfer of Brevibacterium divaricatum DSM 20297T, “Brevibacteriumflavum” DSM 20411, “Brevibacterium lactofermentum” DSM 20412 and DSM1412, and Corynebacterium glutamicum and their distinction by rRNA generestriction patterns. Int. J. Syst. Bacterid. 41:255-260. (1991); andKazuo Komagata et al., “Classification of the coryneform group ofbacteria”. Fermentation and Industry, 45:944-963 (1S87)).

Examples of the genus Brevibacterium include Brevibacterium armoniagenes(for example, ATCC6872). The strain is deposited internationally underthe Budapest Treaty and available to the public.

Examples of the genus Arthrobacter include Arthrobacter globiformis (forexample, ATCC8010, ATCC4336, ATCC21056, ATCC31250, ATCC31738 andATCC35698). These strains are deposited internationally under theBudapest Treaty and available to the public.

Examples of the genus Mycobacterium include Mycobacterium bovis (forexample, ATCC1921Q and ATCC27289). These strains are depositedinternationally under the Budapest Treaty and available to the public.

Examples of the genus Micrococcus include Micrococcus freudenreichii(for example, NO. 239 (FERM P-13221)), Micrococcus leuteus (for example,NO. 240 (FERM P-13222)), Micrococcus ureae (for example, IAM1010), andMicrococcus roseus (for example, IF03764).

The coryneform bacteria may be, let alone a wild type, a mutant thereofor an artificial recombinant thereof. Examples thereof includedisruptants in which a gene of lactate dehydrogenase,phosphoenoipyruvate carboxylase, or malate dehydrogenase is disrupted.Among them, preferred is a disruptant in which a lactate dehydrogenasegene is disrupted. In the disruptant, the lactate dehydrogenase gene isdisrupted and the metabolic pathway from pyruvic acid to lactic acid isblocked. Particularly preferred is a disruptant of Corynebacteriumglutamicum, especially the R (FERM BP-18976) strain in which the lactatedehydrogenase gene is disrupted.

Such a disruptant can be prepared based on a conventional geneengineering process. Such a lactate dehydrogenase disruptant and thepreparation process thereof are described in WO 2005/010182 A1, forexample.

The inventors found that, as shown in FIG. 1, coryneform bacteria haveextremely higher 4-HBA resistance compared with other bacteria. Comparedwith other aerobic bacteria, coryneform bacteria are more resistant tolysis. In this regard, coryneform bacteria are suitable for the 4-HBAproduction by the method of the present invention.

Construction of Vector for Transformant

The DNA which encodes chorismate-pyruvate lyase is amplified by PCR andthen cloned into a suitable vector which is replicable in a host.

The plasmid vector may be any plasmid vector as long as it comprises agene responsible for autonomously replicating function in a coryneformbacterium. Specific examples of the plasmid vector include pAM330 ofBrevibacterium lactofermentum 2256 (JP 58-67696 A; Miwa, K. et al.,Cryptic plasmids in glutamic acid-producing bacteria. Agric. Biol. Chem.48: 2501-2903 (1584;-; and Yamaguchi, R. et al., Determination of thecomplete nucleotide sequence of the Brevibacterium lactofermentumplasmid pAM 330 and the analysis of its genetic information. NucleicAcids Symp. Ser. 16: 265-267 (1985)), pHM1519 of Corynebacteriumglutamicum ATCC3058 (Miwa, K. et al., Cryptic plasmids in glutamicacid-producing bacteria. Agric. Biol. Chem. 48:2901-2903 (1584)), pCRY3Qof the same Corynebacterium glutamicum ATCC3058 (Kurusu, Y. et al.,Identification of plasmid partition function in coryneform bacteria.Appl. Environ. Microbiol. 57: 755-764 (1591)), pCG4 of Corynebacteriumglutamicum T250 (JP 57-183795 A; and Katsumata, R. et al., Protoplasttransformation of glutamate-producing bacteria with plasmid DNA. J.Bacteriol., 159: 306-311 (1984)), pAG1, pAG3, pAG14, and pAG50 of thesame Corynebacterium glutamicum T250 (JP 62-166890 A), pEK0, pEC5, andpEKEx1 of the same Corynebacterium glutamicum T250 (Eikmanns, B. J. etal., A family of Corynebacterium glutamicum/Escherichia coli shuttlevectors for cloning, controlled, gene expression, and promoter probing.Gene, 102: 93-93 (1991)), etc.

Examples of a preferred promoter include promoter PgapA as a promoter ofthe glyceraldehyde-3-phosphate dehydrogenase A gene (gapA), promoterPmdh as a promoter of the malate dehydrogenase gene (mdh), and promoterPldhA as a promoter of lactate dehydrogenase A gene (IdhA), all of whichare of Corynebacterium glutamicum R, and inter alia, PgapA is preferred.

Examples of a preferred terminator include terminator rrnB T1T2 ofEscherichia coli rRNA operon, terminator trpA of Escherichia coli, andterminator trp of Brevibacterium lactofermentum, and inter alia,terminator rrnB T1T2 is preferred.

Transformation

As a method of transformation, any publicly known method can be usedwithout limitation. Examples of such a known method include the calciumchloride/rubidium chloride method, the calcium phosphate method,DEAE-dextran transfection, and electroporation. Among them, preferredfor a coryneform bacterium is electroporation, which can be performed bya known method (Kurusu, Y. et al., Electroporation-transformation systemfor Coryneform bacteria by auxotrophic complementation., Agric. Biol.Chem. 54: 443-447 (1990)).

The transformant is cultured using a culture medium usually used forculture of microorganisms. The culture medium may be a natural medium ora synthetic medium containing a carbon source, a nitrogen source,inorganic salts, other nutritional substances, etc.

Examples of the carbon source include carbohydrates and sugar alcoholssuch as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose,arabinose, galactose, starch, molasses, sorbitol and glycerol; organicacids such as acetic acid, citric acid, lactic acid, fumaric acid,maleic acid and gluconic acid; and alcohols such as ethanol andpropanol. Only one kind of these carbon sources or a mixture of two ormore kinds may be used. The concentration of these carbon sources in theculture medium is usually about 0.1 to 10 w/v%.

Examples of the nitrogen source include inorganic or organic ammoniumcompounds, such as ammonium chloride, ammonium sulfate, ammoniumnitrate, and ammonium acetate; urea; aqueous ammonia; sodium nitrate;and potassium nitrate. Nitrogen-containing organic compounds, such ascorn steep liquor, meat extract, peptone, N-Z-amine, proteinhydrolysate, amino acid, etc. may also be used. Only one kind of thesenitrogen sources or a mixture of two or more kinds may be used. Theconcentration of these nitrogen sources in the culture medium variesdepending on the kind of the nitrogen compound, but is usually 10 w/v%.

Examples of the inorganic salts include potassium dihydrogen phosphate,dipotassium hydrogenphosphate, magnesium sulfate, sodium chloride,iron(II) nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, andcalcium carbonate. Only one kind of these inorganic salts or a mixtureof two or more kinds may be used. The concentration of the inorganicsalts in the culture medium varies depending on the kind of theinorganic salts, but is usually about 0.1 to 1 w/v%.

Examples of the nutritional substances include, for example, meatextract, peptone, poly peptone, yeast extract, dry yeast, corn steepliquor, skim milk powder, defatted soybean hydrochloric acidhydrolysate, and extract from animals, plants or microorganisms, anddegradation products thereof. The concentration of the nutritionalsubstances in the culture medium is usually about 0.1 to 10 w/v%.Further, vitamins may be added as needed. Examples of the vitaminsinclude biotin, thiamine, pyridoxine, pantothenic acid, inositol,nicotinic acid, etc.

The pH of the culture medium is preferably about 6 to 8.

Preferable examples of the microbial culture medium include A medium(Xnui, M. et al., Metabolic analysis of Corynebacterium glutamicumduring lactate and succinate productions under oxygen deprivationconditions. J. Mol. Microbiol. Biotechnol. 7:182-196 (2004)), BT medium(Omumasaba, C. A. et al., Corynebacterium glutamicumglyceraldehyde-3-phosphate dehydrogenase isoforms with opposite,ATP-dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91-103(2004)), etc. The culture temperature is about 15 to 45° C., and theculture period is about 1 to 7 days.

Disruption or Deletion in Host Chromosomal Gene

In the coryneform bacterium as a host, the 4-hydroxybenzoate hydroxylasegene on the chromosome preferably has a disruption or deletion. Due tothe disruption of 4-hydroxybenzoate hydroxylase, the metabolism of 4-HBAproduced is inhibited, resulting in an improved 4-HBA productivity andreduced by-products.

Replacement of a gene on the chromosome with the corresponding genehaving a disruption or deletion can be achieved by creating a gene withdeletion mutation for not producing a normally functioning enzymeprotein, and transforming a bacterium with a DNA comprising the mutatedgene for homologous recombination between the gene on the chromosome andthe mutated gene. An enzyme protein encoded by a gene having adisruption or deletion, even when produced, has a conformation differentfrom that of the wild type, and has no or reduced function. The genedeletion or gene disruption by way of gene substitution through the useof homologous recombination has already been established, and examplesthereof include a method using a plasmid containing a temperaturesensitive replication origin or a plasmid capable of conjugal transfer,and a method using a suicide vector not having a replication origin in ahost (U.S. Pat. No. 6,303,383, JP 05-007491 A).

(3) Process for Producing 4-HBA

4-HBA can be produced by a method comprising a step of reacting thetransformant of the present invention described above in a reactionmixture containing at least one starting compound selected from thegroup consisting of a sugar, a compound that can be metabolized intochorismic acid by the transformant, chorismic acid, and a salt thereof,and a step of recovering 4-HBA from the reaction mixture.

The starting compound must be a compound that can be taken into thetransformant and that is easily available for industrial applications,i.e., one abundantly present in plants, for example.

Glucose is preferred as the sugar, but other sugars that are metabolizedinto glucose can also be used. Such sugars include oligosaccharides andpolysaccharides that have a glucose unit. Examples of such sugarsinclude monosaccharides, such as fructose, mannose, arabinose, xylose,and galactose; disaccharides, such as cellobiose, sucrose, lactose,maltose, trehalose, cellobiose, and xylobiose; polysaccharides, such asdextrin and soluble starch; etc.

Examples of the compound that can be metabolized into chorismic acidinclude quinic acid, shikimic acid, and the like.

Also, molasses, which contains these starting compounds, can also beused, for example. In addition, a saccharified solution which isobtainable by saccharifying, using a diastatic enzyme, non-edibleagricultural waste including straw (rice straw, barley straw, wheatstraw, rye straw, oat straw, etc.), bagasse, and corn stover; energycrops including switchgrass, napier grass, and Miscanthus; wood waste;waste paper; etc. and which contains two or more kinds of sugars,including glucose, can also be used. Among the above-mentioned startingcompounds, glucose, chorismic acid, quinic acid, and shikimic acid arepreferred.

Growth of Microorganism

Before the reaction, the transformant is preferably cultured and grownunder aerobic conditions at about 25 to 38° C. for about 12 to 48 hours.

Culture Medium

The culture medium used for aerobic culture of the transformant beforethe reaction may be a natural medium or a synthetic medium containing acarbon source, a nitrogen source, inorganic salts, other nutritionalsubstances, etc.

Examples of the carbon source that can be used include sugars(monosaccharides such as glucose, fructose, mannose, xylose, arabinose,and galactose; disaccharides such as sucrose, maltose, lactose,cellobiose, xylobiose, and trehalose; polysaccharides such as starch;and molasses); sugar alcohols such as mannitol, sorbitol, xylitol, andglycerol; organic acids such as acetic acid, citric acid, lactic acid,fumaric acid, maleic acid and gluconic acid; alcohols such as ethanoland propanol; and hydrocarbons such as normal paraffin.

Only one kind of these carbon sources or a mixture of two or more kindsmay be used.

Examples of the nitrogen source that can be used include inorganic ororganic ammonium compounds, such as ammonium chloride, ammonium sulfate,ammonium nitrate, and ammonium acetate; urea; aqueous ammonia; sodiumnitrate; and potassium nitrate. Nitrogen-containing organic compounds,such as corn steep liquor, meat extract, peptone, N-Z-amine, proteinhydrolysate, amino acid, etc. may also be used. Only one kind of thesenitrogen sources or a mixture of two or more kinds may be used. Theconcentration of these nitrogen sources in the culture medium variesdepending on the kind of the nitrogen compound, but is usually about 0.1to 10 w/v%.

Examples of the inorganic salts include potassium dihydrogen phosphate,dipotassium hydrogenphosphate, magnesium sulfate, sodium chloride,iron(II) nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, andcalcium carbonate. Only one kind of these inorganic salts or a mixtureof two or more kinds may be used. The concentration of the inorganicsalts in the culture medium varies depending on the kind of theinorganic salts, but is usually about 0.01 to 1 w/v%.

Examples of the nutritional substances include meat extract, peptone,poly peptone, yeast extract, dry yeast, corn steep liquor, skim milkpowder, defatted soybean hydrochloric acid hydrolysate, and extract fromanimals, plants or microorganisms, and degradation products thereof. Theconcentration of the nutritional substances in the culture medium variesdepending on the kind of the nutritional substances, but is usuallyabout 0.1 to 10 w/v%.

Further, vitamins may be added as needed. Examples of the vitaminsinclude biotin, thiamine (vitamin B1), pyridoxine (vitamin B6),pantothenic acid, inositol, nicotinic acid, etc.

The pH of the culture medium is preferably about 6 to 8.

Specific examples of the preferable culture medium for coryneformbacteria include A medium (Inui, M. et al., Metabolic analysis ofCorynebacterium glutamicum during lactate and succinate productionsunder oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol.7:182-196 (2004)), BT medium (Omumasaba, C. A. et al., Corynebacteriumglutamicumglyceraldehyde-3-phosphate dehydrogenase isoforms withopposite, ATP-dependent regulation. J. Mol. Microbiol. Biotechnol.8:91-103 (2004)), etc. Such a culture medium can be used after preparedso as to contain a sugar at a concentration in the above-mentionedrange.

Reaction Mixture

The reaction mixture may be a natural or synthetic reaction mixturecontaining a carbon source, a nitrogen source, inorganic salts, othernutritional substances, etc.

The carbon source may be one or more of the above-described startingcompounds, or a molasses or a saccharified solution containing suchcompounds. As the carbon source, besides sugars, sugar alcohols such asmannitol, sorbitol, xylitol, and glycerol; organic acids such as aceticacid, citric acid, lactic acid, fumaric acid, maleic acid and gluconicacid; alcohols such as ethanol and propanol; and hydrocarbons such asnormal paraffin can also be used.

Only one kind of these carbon sources or a mixture of two or more kindsmay be used.

The concentration of the starting compound in the reaction mixture ispreferably about 1 to 20 w/v%, more preferably about 2 to 10 w/v%, andstill more preferably about 2 to 5 w/v%.

The total concentration of the carbon sources including the startingcompound in the reaction mixture is usually about 2 to 5 w/v%.

Examples of the nitrogen source that can be used include inorganic ororganic ammonium compounds, such as ammonium chloride, ammonium sulfate,ammonium nitrate, and ammonium acetate; urea; aqueous ammonia; sodiumnitrate; and potassium nitrate. Nitrogen-containing organic compounds,such as corn steep liquor, meat extract, peptone, N-Z-amine, proteinhydrolysate, amino acid, etc. may also be used. Only one kind of thesenitrogen sources or a mixture or two or more kinds may be used. Theconcentration of these nitrogen sources in the reaction mixture variesdepending on the kind of the nitrogen compound, but is usually about 0.1to 10 w/v%.

Examples of the inorganic salts include potassium dihydrogen phosphate,dipotassium hydrogenphosphate, magnesium sulfate, sodium chloride,iron(II) nitrate, manganese sulfate, zinc sulfate, cobalt sulfate, andcalcium carbonate. Only one kind of these inorganic salts or a mixtureof two or more kinds may be used. The concentration of the inorganicsalts in the reaction mixture varies depending on the kind of theinorganic salts, but is usually about 0.01 to 1 w/v%.

Further, vitamins may be added as needed. Examples of the vitaminsinclude biotin, thiamine (vitamin B1), pyridoxine (vitamin B6),pantothenic acid, inositol, nicotinic acid, etc. The pH of the reactionmixture is preferably about 6 to 8.

Specific examples of the preferable reaction mixture for coryneformbacteria include the above-mentioned BT medium, etc. Such a culturemedium can be used after prepared so as to contain a sugar at aconcentration in the above-mentioned range.

Reaction Conditions

The reaction temperature, that is, the temperature for keeping thetransformant alive during the reaction is preferably about 20 to 50° C.,and more preferably about 25 to 47° C. When the temperature is in theabove range, 4-HBA can be efficiently produced.

The reaction period is preferably about 1 to 7 days, and more preferablyabout 1 to 3 days.

The culture may be a batch process, a fed-batch process, or a continuousprocess. Among them, a batch process is preferred.

The reaction may be performed under aerobic conditions or reducingconditions. The 4-HBA production ability of the transformant of thepresent invention itself is higher under aerobic conditions. However,aerobic conditions favor the growth of the transformant and the startingcompound is consumed for the growth. Accordingly, the 4-HBA productionefficiency is lowered.

Therefore, it is preferred that the reaction is performed under aerobicconditions where the transformant does not grow. In the presentinvention, “does not grow” includes “substantially does not grow” and“hardly grows”. For example, growth of the transformant can be avoidedor inhibited by the use of a reaction mixture that has a deficiency orlimitation in one or more of compounds essential for the growth of themicroorganism, for example, vitamins, such as biotin and thiamine,nitrogen sources, etc.

Under reducing conditions, coryneform bacteria substantially do notgrow, and therefore, the starting compound is not consumed for thegrowth, which leads to a higher 4-HBA production efficiency.

The “reducing conditions” is defined based on the oxidation-reductionpotential of the reaction mixture. The oxidation-reduction potential ofthe reaction mixture is preferably about −200 mV to −500 mV, and morepreferably about −150 mV to −500 mV.

The reducing conditions of the reaction mixture can be simply estimatedusing resazurin indicator (in reducing conditions, decolorization fromblue to colorless is observed). However, for precise measurement, aredox-potential meter (for example, CRP Electrodes made by BROADLEYJAMES) is used.

As a method of preparing a reaction mixture under reducing conditions,any publicly known method can be used without limitation. For example,as a liquid medium for preparation of the reaction mixture, an aqueoussolution for a reaction mixture may be used instead of distillated wateror the like. As reference for preparation of the aqueous solution for areaction mixture, for example, the method for preparing a culture mediumfor strictly anaerobic microorganisms, such as sulfate-reducingmicroorganisms (Pfennig, N. et al.: The dissimilatory sulfate-reducingbacteria, In The Prokaryotes, A Handbook on Habitats, Isolation andIdentification of Bacteria, Ed. by Starr, M. P. et al. Berlin, SpringerVerlag, 926-940, 1981, or Nogeikagaku Jikkensho, Ed. by Kyoto DaigakuNogakubu Nogeikagaku Kyoshitsu, Vol.3, Sangyo Tosho, 1990, Issue 26) maybe used, and such a method provides an aqueous solution under desiredreducing conditions.

Specifically, by treating distillated water or the like with heat orunder reduced pressure for removal of dissolved gases, an aqueoussolution for a reaction mixture under reducing conditions can beobtained. In this case, for removal of dissolved gases, especiallydissolved oxygen, distillated water or the like may be treated underreduced pressure of about 10 mmHg or less, preferably about 5 mmHg orless, more preferably about 3 mmHg or less, for about 1 to 60 minutes,preferably for about 5 to 40 minutes.

Alternatively, by adding a suitable reducing agent (for example,thioglycolic acid, ascorbic acid, cysteine hydrochloride, mercaptoaceticacid, thiol acetic acid, glutathione, sodium sulfide, etc.), an aqueoussolution for a reaction mixture under reducing conditions can beprepared.

These methods may be suitably combined to prepare an effective aqueoussolution for a reaction mixture under reducing conditions.

In the case of a reaction under reducing conditions, it is preferred tomaintain the reducing conditions of the reaction mixture during thereaction. For maintenance of reducing conditions, it is preferred thatoxygen from the outside of the reaction system is prevented to theutmost extent from entering the system. Specific examples of the methodemployed for this purpose include a method comprising encapsulating thereaction system with inert gas, such as nitrogen gas, carbon dioxidegas, etc. In some cases, for allowing the metabolic functions in thecells of the aerobic bacterium of the present invention to workeffectively during the reaction, addition of a solution of variousnutrients or a reagent solution for adjusting and maintaining the pH ofthe reaction system may be needed. In such a case, for more effectiveprevention of oxygen incorporation, it is effective to remove oxygen inthe solutions to be added, in advance.

Recovery of 4-HBA

Through the culture performed in the above manner, 4-HBA is produced inthe reaction mixture. 4-HBA can be recovered by collecting the reactionmixture, and it is also feasible to isolate 4-HBA from the reactionmixture by a known method. Examples of such a known method include thecrystallization method, the membrane separation method, the organicsolvent extraction method, various adsorption methods (using anion-exchange resin, synthetic adsorbent, or the like), etc.

(4) Method for Improving Chorismate-Pyruvate Lyase Activity

The present invention includes a method for improving or enhancingchorismate-pyruvate lyase activity, the method comprising replacing

(A) valine at position 80 in a chorismate-pyruvate lyase (ubiC) fromPantoea ananatis consisting of the amino acid sequence of SEQ ID NO: 1,or

(B) an amino acid in another chorismate-pyruvate lyase, the amino acidbeing at a position enzymologically homologous with that of the abovevaline, with one or more other amino acids.

Chorismate-pyruvate lyases and the method for mutation thereof are asdescribed above.

The present invention encompasses embodiments in which variousconstituent features described above are combined within the technicalscope of the present invention in such a manner that the effect of thepresent invention is exerted.

EXAMPLES

Hereinafter, the present invention will be illustrated in more detail byExamples, but it is not limited thereto. Various modifications can bemade within the technical idea of the present invention by those withordinary skill in the art.

Example 1 Cloning of 4-HBA-Producing Gene and Construction of ExpressionSystems (Wild Type And Mutants)

(1) Extraction of Chromosomal DNA from Pantoea ananatis

To extract chromosomal DNA from Pantoea ananatis LMG 20103, thebacterium was inoculated into LMG Bacteria Culture Medium No. 1 (1 g ofbeef extract, 2 g of yeast extract, 5 g of peptone, and 5 g of NaCl weredissolved in 1 L of distilled water, and the pH was adjusted to 7.4)using a platinum loop, and cultured with shaking at 28° C. until thelogarithmic growth phase. After the bacterial cells were collected,chromosomal DNA was recovered from the collected cells using a DNAextraction kit (trade name: GenomicPrep Cells and Tissue DNA IsolationKit, made by Amersham) according to the instruction manual.

(2) Cloning of 4-HBA-Producing Gene of Pantoea ananatis

A DNA fragment comprising the ubiC gene which encodes a 4-hydroxybenzoicacid-producing gene (chorismate-pyruvate lyase gene) was amplified bythe PCR method as described below.

In the PCR, the set of primers shown below was synthesized based on SEQID NO: 10 (Pantoea ananatis ubiC gene), and used for cloning of the ubiCgene.

Primers for Pantoea ananatis ubiC gene amplifica- tion (SEQ ID NO: 19)(a-1); 5′-CTCTCATATGACGCAAGACCCGCT-3′ (SEQ ID NO: 20)(b-1); 5′-CTCTCATATGTTAACCTTGATCACGATAGAGCG-3′

Primers (a-1) and (b-1) each have an NdeI restriction enzyme site addedthereto.

Actual PCR was performed using a Veriti thermal cycler (made by AppliedBiosystems) and PrimeSTAR GXL DNA Polymerase (made by Takara Bio, Inc.)as a reaction reagent under the conditions described below.

Reaction Mixture:

PrimeSTAR GXL DNA  1 μL Polymerase (1.24 U/μL) 5× PrimeSTAR GXL Buffer10 μL (Mg²⁺ plus) dNTP Mixture (2.5 mM each)  4 μL Template DNA  1 μL(DNA content:  1 μg or less) The above 2 primers*⁾  1 μL each (finalconc.: 0.2 μM) Sterile distilled water 32 μL

The above ingredients were mixed, and 50 μL of the reaction mixture wassubjected to PCR.

PCR Cycle:

Denaturation step: 98° C., 10 seconds

Annealing step: 50° C., 5 seconds

Extension step: 68° C., 31 seconds

A cycle consisting of the above 3 steps was repeated 30 times.

Using 10 μL of the above-produced reaction mixture, 0.8% agarose gelelectrophoresis was performed, and an about 0.5-kb DNA fragment of theubiC gene of Pantoea ananatis was detected. The DNA fragment waspurified using NucleoSpin Gel and PCR Clean-Up (made by Takara Bio,Inc.).

(3) Construction of 4-hydroxybenzoic Acid-Producing Gene(Chorismate-Pyruvate Lyase Gene) Expression Plasmid

10 μL of the about 0.5-kb DNA fragment comprising the ubiC gene ofPantoea ananatis amplified by the PCR in the above (2) and 2 μL of thecloning vector pCRB209 (WO 2012/033112) comprising a promoter PgapA wereeach cut with the use of restriction enzyme NdeI and processed at 70° C.for 10 minutes for deactivation of the restriction enzyme. Both weremixed, and 1 μL of T4 DNA ligase 10× buffer solution and 1 unit of T4DNA ligase (made by Takara Bio, Inc.) were added thereto. Steriledistilled water was added thereto so that the total volume, was 10 μL,and the mixture was allowed to react at 1.5° C. for 3 hours forligation.

Using the obtained ligation liquid, Escherichia coli HST02 wastransformed by the calcium chloride method (Journal of MolecularBiology, 53, 159 (1970)) and was applied to LB agar medium (1%polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar)containing 50 μg/mL of kanamycin.

A growing strain on the culture medium was subjected to liquid culturein the usual manner. Plasmid DNA was extracted from the culture mediumand cut using the restriction enzyme, to confirm the inserted fragment.As a result, in addition to an about 5.1-kb DNA fragment of the plasmidpCRB209, an about 0.5-kb inserted fragment of the ubiC gene of Pantoeaananatis was confirmed.

The plasmid comprising the ubiC gene of Pantoea ananatis was namedpH3A22.

(4) Construction of Transgenic Strains for 4-hydroxybenzoicAcid-Producing Gene (Chorismate-Pyruvate Lyase Gene)

Using the above-described plasmid pHBA22, transformation ofCorynebacterium glutamicum R was performed by electroporation [Agric.Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144,181-185 (1993)], and the transgenic strain was applied to A agar mediumcontaining 50 μg/mL of kanamycin.

A growing strain on the culture medium was subjected to liquid culturein the usual manner. Plasmid DNA was extracted from the culture and cutwith the use of a restriction enzyme to confirm the inserted plasmid. Asa result, introduction of the above-constructed plasmid pHBA22 wasconfirmed.

The obtained strain was named Corynebacterium glutamicum HBA-22.

(5) Construction of Transgenic Plasmids for 4-hydroxybenzoicAcid-Producing Genes (Chorismate-Pyruvate Lyase Genes) with Mutation bySite-Directed Mutagenesis

Using the above-described plasmid pHBA22, two mutants having differentkinds of amino acids in place of the amino acid at the V80 site wereprepared by Inverse PCR, and the obtained site-specific transgenicplasmids were named pHBA23 and pH3A24.

In the PCR, the set of primers shown below was synthesized based on SEQID NO: 10 (Pantoea ananatis ubiC gene), and used for introduction ofmutation to the V80 site of the ubiC gene.

Primers for mutation of Pantoea ananatis ubiC      gene (SEQ ID NO: 21)(a-2); 5′-CGAGAAgcaATTCTCTACGGGGATG-3′ (SEQ ID NO: 22)(b-2); 5′-CAGCCAGAAACGCTGATCG-3′ (SEQ ID NO: 23)(a-3); 5′-tgcATTCTCTACGGGGATGAGG-3′ (SEQ ID NO: 24)(b-3); 5′-TTCTCGCAGCCAGAAACGCTG-3′

Actual PCR was performed using a Veriti thermal cycler (made by AppliedBiosystems) and PrimeSTAR GXL DNA Polymerase (made by Takara Bio, Inc.)as a reaction reagent under the conditions described below.

Reaction Mixture:

PrimeSTAR GXL Data Polymerase  1 μL (1.25 U/μL) 5× PrimeSTAR GXL Buffer10 μL (Mg²⁺ plus) dNTP Mixture (2.5 mM each)  4 μL Template DNA  1 μL(DNA content: 1 μg or less) The above 2 primers*⁾  1 μL each (finalconc.: 0.2 μM) Sterile distilled water 32 μL The above ingredients weremixed, and 50 μL of the reaction mixture was subjected to PCR. *⁾Foramplification of pHBA23 (V80A), a combination of primers (a-2) and(b-2), and for amplification of pHBA24 (V80C), a combination of primers(a-3) and (b-3) were used.

PCR Cycle:

Denaturation step: 98PC, 10 seconds

Annealing step: 50° C., 5 seconds

Extension step: 68° C., 338 seconds

A cycle consisting of the above 3 steps was repeated 30 times.

Using 10 μL each of the above-produced reaction mixtures, 0.8% agarosegel electrophoresis was performed, and an about 5.6-kb DNA fragment ofthe ubiC gene of Pantoea ananatis was detected in both cases. The DNAfragment was purified using NucleoSpin Gel and PCR Clean-Up (made byTakara Bio, Inc.).

The purified amplification product was phosphorylated using T4Polynucleotide Kinase (made by Takara Bio, Inc.) and then purified usingNucleoSpin Gel and PCR Clean-Up (made by Takara Bio, Inc.). The obtainedphosphorylated DNA fragment was allowed to self-ligate using the DNALigation Kit (made by Takara Bio, Inc.). Using the obtained ligationliquid, Escherichia coli HST02 was transformed by the calcium chloridemethod (J. Mol. Biol. 53: 159-162 (1970)) and was applied to LB agarmedium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and1.5% agar) containing 50 μg/mL of kanamycin. A growing strain on theculture medium was subjected to liquid culture in the usual manner.Plasmid DNA was extracted from the culture, and the introduction of themutation into the V80 of the ubiC gene was confirmed by the sequenceanalysis of the plasmid.

The obtained plasmids were named pHBA23 and pHBA24. The outline of generecombination of the plasmids is shown in Table 1.

TABLE 1 Transgenic plasmids (wild-type and mutants) for 4-HBA-producinggene of Pantoea ananatis Type of introduced Amino acid Codon Plasmidmutation at position 80 at position 80 pHBA22 None (wild type) valinegtc pHBA23 V80A alanine gca pHBA24 V80C cysteine tgc(6) Construction of Transgenic Strains for 4-hydroxybenzoicAcid-Producing Gene (Chorismate-Pyruvate Lyase Gene)

Using the above-described plasmids pHBA22 to pHBA24, transformation ofCorynebacterium glutamicum R was performed by electroporation (Agric.Biol. Chem., Vol. 54, 443-447 (1990) and Res. Microbiol., Vol. 144,181-185 (19S3)), and each of the transgenic strains was applied to Aagar medium containing 50 Hg/mL of kanamycin.

A growing strain on the culture medium was subjected to liquid culturein the usual manner. Plasmid DNA was extracted from the culture and cutwith the use of a restriction enzyme to confirm the inserted plasmid. Asa result, introduction of the above-constructed plasmids pHBA22 topHBA24 was confirmed.

The obtained strains were named Corynebacterium glutamicum HBA-22 toHBA-24. The outline of gene recombination in the above-obtained strainsis shown in Table 2.

TABLE 2 Transgenic strains for 4-HBA-producing gene (mutant) of Pantoeaananatis Type of introduced Amino acid Strain Host strain mutation atposition 80 HBA22 Corynebacterium None (wild type) valine HBA23glutamicum R V80A alanine HBA24 V80C cysteine

Example 2

Comparison of Chorismate-Pyruvate Lyase Activity Among Corynebacteriumglutamicum 4-hydroxybenzoic Acid-Producing Gene Transgenic Strains

Using the cell lysates obtained by sonication of Corynebacteriumglutamicum KBA-22 (V80, wild type), HBA-23 (V80A), and HBA-24 (V80C)prepared in Example X, comparison of the chorismate-pyruvate lyaseactivity was performed.

Specifically, each of the above strains was applied to A agar medium (2g of (NH₂)₂CO, 7 g of (NH₄)₂SO₄, 0.5 g of KH₂PO_(4,) 0.5 g of K₂HPO₄,0.5 g of MgSO₄.7H₂O, 1 mL of 0.06% (w/v) Fe₂SO₄.7H₂O+0.042% (w/v)MnSO₄.2H₂O, 1 ml of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v)thiamin solution, 2 g of yeast extract, 7 g of vitamin assay casaminoacid, 40 g of glucose, and 15 g of agar were suspended in 1 L ofdistilled water) containing 50 μg/mL of kanamycin and was left stand inthe dark at 33° C .for 15 hours.

An inoculation loop of each strain grown on a plate as above wasinoculated into a test tube containing 10 mL of A liquid medium (2 g of(NH₂)₂CO, 7 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄, 0.5 g of K₂HPO₄, 0.5 g ofMgSO₄.7H₂O, 1 mL of 0.06% (w/v) Fe₂SO₄.7H₂O+0.042% (w/v) MnSO₄.2H₂O, 1ml of 0.02% (w/v) biotin solution, 2 mL of 0.01% (w/v) thiamin solution,2 g of yeast extract, 7 g of vitamin assay casamino acid, and 40 g ofglucose were suspended in 1 L of distilled water) containing 50 μg/mL ofkanamycin and was aerobically cultured with shaking at 33° C. for 15hours.

Each kind of the bacterial cells cultured and proliferated as above wascollected by centrifugation (8,000 rpm, 4° C., 10 minutes). Afterdisrupting the bacterial cells by sonication, centrifugation (15,000rpm, 4° C., 20 minutes) was performed. Using the supernatant of the celllysate as a crude enzyme liquid, chorismate-pyruvate lyase activity wasdetermined by the following method.

The crude enzyme liquid, 50 mM Tris-HCl (pH 7.5), 0.5 mM of chorismateBa salt, 0.2 mM of NADH, 0.2 M of NaCl and 5 units of lactatedehydrogenase were mixed, the reaction was allowed to proceed at 33° C.,and the decrease in absorbance of NADH at 340 nm was monitored toanalyze the initial rate of the reaction. From the initial rate of thereaction and the protein concentration, the specific activity wascalculated (the amount of the enzyme that produces 1 μmol of 4-HBA perminute was defined as 1 unit). (After the reaction mixture was filtered,the resulting 4-HBA was subjected to HPLC for direct detection of thepeak of 4-HBA (Cosmosil C18 ARII made by Nacalai Tesque, mobile phase:20% methanol and 0.07% perchloric acid) to confirm that the two assaymethods were similar in quantitative performance.)

As a result, as shown in Table 3, the V80A mutant and the V80C mutantshowed a higher activity as compared with the V80 (wild type) strain.

TABLE 3 Comparison of chorismate-pyruvate lyase activity amongtransgenic strains for 4-HBA-producing gene (mutant) of Pantoea ananatisType Enzymatic introduced Amino acid activity Strain Host strainmutation at position 80 (mU · mg⁻¹) HBA22 Corynebacterium None valine339 glutamicum R (wild type) HBA23 V80A alanine 485 HBA24 V80C cysteine341

Example 3

Production of 4-hydroxybenzoic Acid From Glucose Using Corynebacteriumglutamicum 4-hydroxybenzoic Acid-Producing Gene Transgenic Strains

Each of the Corynebacterium glutamicum HBA-22 (wild type), HBA-23(V80A), and HBA-24 (V30C) strains prepared in Example 1 was applied to Aagar medium (2 g of (NH₂)₂CO, 7 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄, 0.5 gof K₂HPO₄, 0.5 g of MgSO₄.7H₂O, 1 mL of 0.06% (w/v) Fe₂SO₄.7H₂O+0.042%(w/v) MnSO₄.2H₂O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01%(w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assaycasamino acid, 40 g of glucose, and 15 g of agar were suspended in 1 Lof distilled water) containing 50 μg/mL of kanamycin and was left standin the dark at 33° C. for 15 hours.

An inoculation loop of each of the Corynebacteriumglutamicum/4-HBA-producing gene transgenic strains grown on a plate asabove was inoculated into a test tube containing 10 mL of A liquidmedium (2 g of (NH₂)₂CO, 7 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄, 0.5 g ofK₂HPO₄, 0.5 g of MgSO₄.7H₂O, 1 mL of 0.06% (w/v) Fe₂SO₄.7H₂O+0.042%(w/v) MnSO₄.2H₂O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01%(w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assaycasamino acid, and 40 g of glucose were suspended in 1 L of distilledwater) containing 50 ng/mL of kanamycin and also 2% of calcium carbonateand was aerobically cultured with shaking at 200 rpm at 33° C. for 24hours.

The culture obtained after the growth under the above-describedconditions was centrifuged (15,000 rpm at 4° C. for 10 minutes), and theobtained supernatant was used for quantitative determination of 4-HBA byKPLC.

As a result, as shown in Table 4, the HBA-23 (V80A) and HBA-24 (V80C)strains produced 4-HBA at an about 2.3 times higher concentration and anabout 1.6 times higher concentration, respectively, as compared with theHBA-22 (wild type) strain.

TABLE 4 Experiment of 4-HBA production from glucose usingCorynebacterium glutamicum 4-HBA-producing gene transgenic strains(transgenic strains for ubiC gene of Pantoea ananatis) Origin of Amountof 4-hydroxy produced benzoic 4-hydroxy acid- Type of benzoic acidproducing introduced (mM) Strain Host strain gene mutation (After 24hours) HBA22 Corynebacterium Pantoea None 0.70 glutamicum R ananatis(wild HBA23 V80A 1.64 HBA24 V80C 1.14

Example 4

Extraction of Chromosomal DNA from Providencia bacteria, Cloning of4-hydroxybenzoic Acid-Producing Genes (Chorismate-Pyruvate Lyase Genes),Construction of 4-hydroxybenzoic Acid-Producing Gene ExpressionPlasmids, Construction of 4-hydroxybenzoic Acid-Producing GeneTransgenic Strains, Construction of Site-Specific Transgenic Strains for4-hydroxybenzoic Acid-Producing Genes, And Production of4-hydroxybenzoic Acid from Glucose Using Corynebacterium glutamicumTransgenic Strains for 4-hydroxybenzoic Acid-Producing Genes(1) Extraction of Chromosomal DNA from Microorganisms

To extract chromosomal DNA from Providencia stuartii ATCC 25827, thebacterium was inoculated into ATCC Medium No. 3 (5 g of peptone and 3 gof beef extract were dissolved in 1 L of distilled water, and the pH wasadjusted to 6.8) using a platinum loop, and cultured with shaking at 37°C. until the logarithmic growth phase. After the bacterial cells werecollected, chromosomal DNA was recovered from the collected cells usinga DNA extraction kit (trade name: GenomicPrep Cells and Tissue DNAIsolation Kit, made by Amersham) according to the instruction manual.

To extract chromosomal DNA from Providencia rustigianii JCM 3953, thebacterium was inoculated into JCM Medium No. 12 (5 g of peptone, 3 g ofbeef extract, and 5 g of NaCl were dissolved in 1 L of distilled water,and the pH was adjusted to 7.0) using a platinum loop, and cultured withshaking at 37° C. until the logarithmic growth phase. After thebacterial cells were collected, chromosomal DNA was recovered from thecollected cells using a DNA extraction kit (trade name: GenomicPrepCells and Tissue DNA isolation Kit, made by Amersham) according to theinstruction manual.

To extract chromosomal DNA from Escherichia coli K12 MG1655, thebacterium was inoculated into LB Medium (10 g of tryptone, 5 g of yeastextract, and 5 g of NaCl were dissolved in 1 L of distilled water) usinga platinum loop, and cultured with shaking at 37° C. until thelogarithmic growth phase. After the bacterial cells were collected,chromosomal DNA was recovered from the collected cells using a DNAextraction kit (trade name: GenomicPrep Cells and Tissue DNA IsolationKit, made by Amersham) according to the instruction manual.

To extract chromosomal DNA from Cronobacter sakazakii JCM 1233, thebacterium was inoculated into JCM Medium No. 12 (5 g of peptone, 3 g ofbeef extract, and 5 g of NaCl were dissolved in 1 L of distilled water,and the pH was adjusted to 7.0) using a platinum loop, and cultured withshaking at 37° C. until the logarithmic growth phase. After thebacterial cells were collected, chromosomal DNA was recovered from thecollected cells using a DNA extraction kit (trade name: GenomicPrepCeils and Tissue DNA Isolation Kit, made by Amersham) according to theinstruction manual.

(2) Cloning of 4-hydroxybenzoic Acid-Producing Genes(Chorismate-Pyruvate Lyase Genes)

A DNA fragment comprising the ubiC gene which encodes a 4-hydroxybenzoic acid-producing gene (chorismate-pyruvate lyase gene) wasamplified by the PCR method as described below.

In the PCR, the sets of primers shown below were synthesized based onSEQ ID NO: 11 (Providencia stuartii ubiC gene), SEQ ID NO: 12(Providencia rustigianii ubiC gene), SEQ ID NO: 17 (Escherichia coliubiC gene), and SEQ ID NO: 18 (Cronobacter sakazakii ubiC gene), andused for cloning of. the corresponding ubiC genes.

Primers for Providencia stuartii ubiC gene amplification (SEQ ID NO: 25)(a-21); 5′-CTCTCATATGGATGAAACGCTTTTTATCTCTCAC-3′ (SEQ ID NO: 26)(b-21); 5′-CTCTCATATGTCCCTCCATTTGTTGTGCTC-3′

Primers (a-21) and (b-21) each have an NdeI restriction enzyme siteadded thereto.

Primers for Providencia rustigianii ubiC gene amplification(SEQ ID NO: 27) (a-22); 5′-CTCTCATATGCATGAAACAATTTTTACCCATCATCC-3′(SEQ ID NO: 28) (b-22); 5′-CTCTCATATGGATTATGTTAGATAGTTATCTATATGCAGGTG-3′

Primers (a-22) and (b-22) each have an NdeI restriction enzyme siteadded thereto.

Primers for Escherichia coli ubiC gene amplifica- tion (SEQ ID NO: 29)(a-23); 5′-CTCTCATATGTCACACCCCGCGTTAA-3′ (SEQ ID NO: 30)(b-23); 5′-CTCTCATATGTTAGTACAACGGTGACGCC-3′

Primers (a-23) and (b-23) each have an NdeI restriction enzyme siteadded thereto.

Primers for Cronobacter sakazakii ubiC gene  amplification(SEQ ID NO: 31) (a-24); 5′-CTCTCATATGTCCCATCCCGCGCTGAG-3′(SEQ ID NO: 32) (b-24); 5′-CTCTCATATGTATTCTGCGTCAGGCTCCAC-3′

Primers (a-24) and (b-24) each have an NdeI restriction enzyme siteadded thereto.

As the template DNA, chromosomal DNAs extracted from Providenciarustigianii JCM 3953, Providencia stuartii ATCC 25827, Escherichia coliMG1655, and Cronobacter sakazakii JCM 1233 were used.

Actual PCR was performed using a Veriti thermal cycler (made by AppliedBiosystems) and PrimeSTAR GXL DNA Polymerase (made by Takara Bio, Inc.)as a reaction reagent under the conditions described below.

Reaction Mixture:

PrimeSTAR GXL DNA Polymerase  1 μL (1.25 U/μL) 5× PrimeSTAR GXL Buffer10 μL (Mg²⁺ plus) dNTP Mixture (2.5 mM each)  4 μL Template DNA  1 μL(DNA content: 1 μg or less) The above 2 primers*⁾  1 μL each (finalconc.: 0.2 μM) Sterile distilled water 32 μL The above ingredients weremixed, and 50 μL of the reaction mixture was subjected to PCR. *⁾Foramplification of the ubiC gene of Providencia stuartii, a combination ofprimers (a-21) and (b-21); for amplification of the ubiC gene ofProvidencia rustigianii, a combination of primers (a-22) and (b-22); foramplification of the ubiC gene of Escherichia coli, a combination ofprimers (a-23) and (b-23); and for amplification of the ubiC gene ofCronobacter sakazakii, a combination of primers (a-24) and (b-24) wereused.

PCR Cycle:

Denaturation step: 98° C., 10 seconds

Annealing step: 50° C., 5 seconds

Extension step: 68° C.

-   -   Providencia stuartii ubiC gene, 32 seconds    -   Providencia rustigianii ubiC gene, 31 seconds    -   Escherichia coli ubiC gene, 30 seconds    -   Cronobacter sakazakii ubiC gene, 32 seconds

A cycle consisting of the above 3 steps was repeated 30 times.

Using 10 μL each of the above-produced reaction mixtures, 0.8% agarosegel electrophoresis was performed. As a result, detected were an about0.5-kb DNA fragment in the case of the ubiC gene of Providenciastuartii, in the case of the ubiC gene of Providencia rustigianii, andin the case of the ubiC gene of Escherichia coli; and an about 0.6-kbDNA fragment in the case of the ubiC gene of Cronobacter sakazakii. EachDNA fragment was purified using NucleoSpin Gel and PCR Clean-Up (made byTakara Bio, Inc.).

(3) Construction of 4-hydroxybenzoic Acid-Producing Gene(Chorismate-Pyruvate Lyase Gene) Expression Plasmids

10 μL of the about 0.5-kb DNA fragment comprising the ubiC gene ofProvidencia stuartii, the ubiC gene of Providencia rustigianii, or theubiC gene of Escherichia coli; or the about 0.6-kb DNA fragmentcomprising the ubiC gene of Cronobacter sakazakii, each of which wasamplified by the PCR as above, and 2 μL of the cloning vector pCRB209comprising a promoter PgapA (WO 2012/033112) were each cut using therestriction enzyme NdeI and processed at 70° C. for 10 minutes fordeactivation of the restriction enzyme. Both were mixed, and 1 μL of T4DNA ligase 10× buffer solution and 1 unit of T4 DNA ligase (made byTakara Bio, Inc.) were added thereto. Sterile distilled water was addedthereto so that the total volume was 10 μL, and the mixture was allowedto react at 15° C. for 3 hours for ligation.

Using the obtained ligation liquids separately, Escherichia coli HST02was transformed by the calcium chloride method (Journal of MolecularBiology, 53, 159 (1970)) and was applied to LB agar medium (1%polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and 1.5% agar)containing 50 fig/mL of kanamycin.

A growing strain on the culture medium was subjected to liquid culturein the usual manner. Plasmid DNA was extracted from the culture and cutusing the restriction enzyme to confirm the inserted fragment. As aresult, in addition, to an about 5.1-kb DNA fragment of the plasmidpCRB209, confirmed were an about 0.5-kb inserted fragment in the case ofthe ubiC gene of Providencia stuartii, an about 0.5-kb inserted fragmentin the case of the ubiC gene of Providencia rustigianii, an about 0.5-kbDNA fragment in the case of the ubiC gene of Escherichia coli, and anabout 0.6-kb DNA fragment in the case of the ubiC gene of Cronobactersakazakii.

The plasmid comprising the ubiC gene of Providencia stuartii was namedpHBA42, the plasmid comprising the ubiC gene of Providencia rustigianiiwas named pHBA45, the plasmid comprising the ubiC gene of Escherichiacoli was named pH8A48, and the plasmid comprising the ubiC gene ofCronobacter sakazakii was named pHBA51.

(4) Construction of Transgenic Plasmids for 4-hydroxybenzoicAcid-Producing Genes (Chorismate-Pyruvate Lyase Genes) with Mutation bySite-Directed Mutagenesis

Using the above-described plasmid pHBA42, mutants in which the aminoacid at the V80 site was replaced with A (alanine) or C (cysteine) wereprepared by Inverse PCR, and the obtained site-specific transgenicplasmids were named pHBA43 and pHBA44 .

In the PCR, the set of primers shown below was synthesized based on SEQID NO: 11 (Providencia stuartii ubiC gene), and used for introduction ofmutation to the V80 site of the ubiC gene.

Primers for mutation of Providencia stuartii ubiC gene (SEQ ID NO: 33)(a-25); 5′-gcaATTATGTATGGTGATAATATTCCATGGTTACTTG-3′ (SEQ ID NO: 34)(a-26); 5′-tgcATTATGTATGGTGATAATATTCCATGGTTACTTG-3′ (SEQ ID NO: 35)(b-25); 5′-TTCACGTAACCAATAATATTCACTGACAG-3′

Similarly, using the above-described plasmid pHBA45, mutants in whichthe amino acid at the V80 site was replaced with A (alanine) or C(cysteine) were prepared by Inverse PCR, and the obtained site-specifictransgenic plasmids were named pHBA4 6 and pHBA47.

In the PCR, the set of primers shown below was synthesized based on SEQID NO: 12 (Providencia rustigianii ubiC gene), and used for introductionof mutation to the V80 site of the ubiC gene.

Primer for mutation of Providencia rustigianii ubiC gene (SEQ ID NO: 36)(a-27); 5′-ATTATGTATGGGGATAATATTCCGTGG-3′ (SEQ ID NO: 37)(b-27); 5′-gcaTTCTCGCAACCAGTAACGTTG-3′ (SEQ ID NO: 38)(b-28); 5′-tgcTTCTCGCAACCAGTAACGTTG-3′

In the PCR, the set of primers shown below was synthesized based on SEQID NO: 17 (Escherichia coli ubiC gene), and used for introduction ofmutation to the 1 (isoleucine) 79 site of the ubiC gene.

Primers for mutation of Escherichia coli ubiC gene (SEQ ID NO: 39)(a-29); 5′-gcaTTGTTATGTGCCGATGGTGAAC-3′ (SEQ ID NO: 40)(a-30); 5′-tgcTTGTTATGTGCCGATGGTGAAC-3′ (SEQ ID NO: 41)(b-29); 5′-TTCACGTAACCAATAATATTCACTGACAG-3′

Similarly, using the above-described plasmid pHBA51, mutants in whichthe amino acid at the I (isoleucine) 79 site was replaced with A(alanine) or C (cysteine) were prepared by PCR, and the obtainedsite-specific transgenic plasmids were named pHBA52 and pHBA53.

In the PCR, the set of primers shown below was synthesized based on SEQID NO: 18 (Cronobacter sakazakii ubiC gene), and used for introductionof mutation to the 179 site of the ubiC gene.

Primers for mutation of Cronobacter sakazakii  ubiC gene (SEQ ID NO: 42)(a-31); 5′-gcaCTGCTGTGCGGCGACG-3′ (SEQ ID NO: 43)(a-32); 5′-tgcCTGCTGTGCGGCGACG-3′ (SEQ ID NO: 44)(b-31); 5′-TTCGCGCAGCCAGTAGCG-3′

Actual PCR was performed using a Veriti thermal cycler (made by AppliedBiosystems) and PrimeSTAR GXL DNA Polymerase (made by Takara Bio, Inc.)as a reaction reagent under the conditions described below.

Reaction Mixture:

PrimeSTAR GXL DNA Polymerase  1 μL (1.25 U/μL) 5× PrimeSTAR GXL Buffer10 μL (Mg²⁺ plus) dNTP Mixture (2.5 mM each)  4 μL Template DNA  1 μL(DNA content: 1 μg or less) The above 2 primers*⁾  1 μL each (finalconc.: 0.2 μM) Sterile distilled water 32 μL The above ingredients weremixed, and 50 μL of the reaction mixture was subjected to PCR. *⁾Foramplification of pHBA43, a combination of primers (a-25) and (b-25); foramplification of pHBA44, a combination of primers (a-26) and (b-25); foramplification of pHBA46, a combination of primers (a-27) and (b-27); foramplification of pHBA47, a combination of primers (a-27) and (b-28); foramplification of pHBA49, a combination of primers (a-29) and (b-29); foramplification of pHBA50, a combination of primers (a-30) and (b-29); foramplification of pHBA52, a combination of primers (a-31) and (b-31); andfor amplification of pHBA53, a combination of primers (a-32) and (b-31)were used.

PCR Cycle:

Denaturation step: 98° C., 10 seconds

Annealing step: 50° C., 5 seconds

Extension step: 68° C.

-   -   Providencia stuartii (pHBA43, pHBA44), 339 seconds    -   Providencia rustigianii (pHBA46, pHBA47), 338 seconds    -   Escherichia coli (pHBA49, pHBA50), 337 seconds    -   Cronobacter sakazakii (pHBA52, pHBA53), 339 seconds

A cycle consisting of the above 3 steps was repeated 30 times.

Using 10 μL each of the above-produced reaction mixtures, 0.8% agarosegel electrophoresis was performed. As a result, detected were an about5.7-kb DNA fragment in the case of the ubiC gene of Providenciastuartii, an about 5.6-kb DNA fragment in the case of the ubiC gene ofProvidencia rustigianii, an about 5.6-kb DNA fragment in the case of theubiC gene of Escherichia coli, and an about 5.7-kb DNA fragment in thecase of the ubiC gene of Cronobacter sakazakii. Each DNA fragment waspurified using NucleoSpin Gel and PCR Clean-Up (made by Takara Bio,Inc.).

The purified amplification product was phosphorylated using T4Polynucleotide Kinase (made by Takara Bio, Inc.) and then purified usingNucleoSpin Gel and PCR Clean-Up (made by Takara Bio, Inc.). The obtainedphosphorylated DNA fragment was allowed to self-ligate using the DNALigation Kit (made by Takara Bio, Inc.). Using the obtained ligationliquid, Escherichia coli HST02 was transformed by the calcium chloridemethod (J. Mol. Biol. 53: 159-162 (1970)) and was applied to LB agarmedium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride, and1.5% agar) containing 50 μg/mL of kanamycin. A growing strain on theculture medium was subjected to liquid culture in the usual manner.Plasmid DNA was extracted from the culture, and the introduction of themutation into the V80 or 179 site of the ubiC gene was confirmed by thesequence analysis of the plasmid.

The obtained plasmids were named pHBA43, pHBA44, pHBA46, PHBA47, pHBA49,pHBA50, pHBA52, and pHBA53. The outline of gene recombination of theplasmids is shown in Table 5.

TABLE 5 Transgenic plasmids for 4-HBA-producing gene (WT and mutants)Origin of ubiC Codon Plasmid gene Type of introduced mutation* usedpHBA42 Providencia Wild type (V80) gtc pHBA43 stuartii V80A gca pHBA44V80C tgc pHBA45 Providencia Wild type (V80) gtc pHBA46 rustigianii V80Agca pHBA47 V80C tgc pHBA48 Escherichia coli Wild type (I79) att pHBA49I79A gca pHBA50 I79C tgc pHBA51 Cronobacter Wild type (I79) atc pHBA52sakazakii I79A gca pHBA53 I79C tgc In the column of the “Type ofintroduced mutation*”, A means “a mutation to alanine”, and C means “amutation to cysteine”. Also, V means “a mutation of valine”, and I means“a mutation of isoleucine”. In addition, 80 means “position 80” and 79means “position 79”.(5) Construction of Transgenic Strains for 4-hydroxybenzoicAcid-Producing Gene (Chorismate-Pyruvate Lyase Gene)

Using the above-described plasmids pHBA42, pHBA43, pHBA44, pHBA45,pHBA46, pHBA47, pHBA48, pHBA49, pHBA50, pHBA51, pHBA52, and pHBA53,transformation of Corynebacterium glutamicum R was performed byelectroporation [Agric. Biol. Chem., Vol. 54, 443-447 (1590) and Res.Microbiol., Vol. 144, 181-185 (1993)], and each of the transgenicstrains was applied to A agar medium containing 50 μg/mL of kanamycin.

A growing strain on the culture medium was subjected to liquid culturein the usual manner. Plasmid DNA was extracted from the culture and cutwith the use of a restriction enzyme to confirm the inserted plasmid. Asa result, introduction of the above-prepared plasmids pHBA42, pHBA43,pHBA44, pHBA45, pHBA46, pHBA47, pHBA48, pHBA49, pHBA50, pHBA51, pHBA52,and pHBA53 was confirmed.

The obtained strains were named Corynebacterium glutamicum HBA42, HBA43,HBA44, HBA45, HBA46, HBA47, HBA48, HBA45, HBA50, HBA51, HBA52, andHBA53. The outline of gene recombination of the plasmids is shown inTable 6.

TABLE 6 Transgenic strains for 4-HBA-producing gene (mutant) Type ofOrigin of introduced Strain Host strain Plasmid ubiC gene mutation*HBA42 Corynebacterium pHBA42 Providencia Wild type HBA43 glutamicum RpHBA43 stuartii V80A HBA44 pHBA44 V80C HBA45 pHBA45 Providencia Wildtype HBA46 pHBA46 rustigianii V80A HBA47 pHBA47 V80C HBA48 pHBA48Escherichia Wild type HBA49 pHBA49 coli I79A HBA50 pHBA50 I79C HBA51pHBA51 Cronobacter Wild type HBA52 pHBA52 sakazakii I79A HBA53 pHBA53I79C In the column of the “Type of introduced mutation*”, A means “amutation to alanine”, and C means “a mutation to cysteine”. Also, Vmeans “a mutation of valine”, and I means “a mutation of isoleucine”. Inaddition, 80 means “position 80” and 79 means “position 79”.

Each of the Corynebacterium glutamicum/4-HBA-producing gene transgenicstrains obtained as above (HBA42, HBA43, HBA44, HBA45, HBA46, HBA47,HBA48, HBA49, HBA50, HBA51, HBA52, and HSA53) was applied to A agarmedium (2 g of (NH₂)₂CO, 7 g of (NR₄)₂SO₄, 0.5 g of KH₂PO₄, 0.5 g ofK₂HPO₄, 0.5 g of MgSO₄.7H₂O, 1 mL of 0.06% (w/v) Fe₂SO₄.7H₂O+0.042%(w/v) MnSO₄.2H₂O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.0.1%(w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assaycasamino acid, 40 g of glucose, and 15 g of agar were suspended in 1 Lof distilled water) containing 50 μg/mL of kanamycin and was left standin the dark at 33° C. for 15 hours.

An inoculation loop of each of the Corynebacteriumglutamicum/4-HBA-producing gene transgenic strains grown on a plate asabove was inoculated into a test tube containing 10 mL of A liquidmedium (2 g of (NH₂)₂CO, 7 g of (NH₄)₂SO₄, 0.5 g of KH₂PO₄, 0.5 g ofK₂HPO₄, 0.5 g of MgSO₄.7H₂O, 1 mL of 0.06% (w/v) Fe₂SO₄.7H₂O+0.042%(w/v) MnSO₄.2H₂O, 1 mL of 0.02% (w/v) biotin solution, 2 mL of 0.01%(w/v) thiamin solution, 2 g of yeast extract, 7 g of vitamin assaycasamino acid, and 40 g of glucose were suspended in 1 L of distilledwater) containing 50 μg/mL of kanamycin and also 2% of calcium carbonateand was aerobically cultured with shaking at 33° C. for 24 hours.

The culture obtained after the growth under the above-describedconditions was centrifuged (15,000 rpm at 4° C. for 10 minutes), and theobtained supernatant was used for quantitative determination of 4-HBA byKPLC.

As the results in Table 7 show, each of the strains of Corynebacteriumglutamicum HBA-42 to 53 produced the aimed 4-HBA. Among the transgenicstrains for ubiC gene from Providencia stuartii, HBA-43 (V80A) andHBA-44 (V30C) had an enhanced, i.e., superior 4-HBA-producing ability,as compared with HBA-42 (wild type). Among the transgenic strains forubiC gene from Providencia rettgeri, HBA-46 (V80A) and HBA-47 (V80C) hadan enhanced, i.e., superior 4-HBA-producing ability, as compared withHBA-45 (wild type). Among the transgenic strains for ubiC gene fromEscherichia coli, HSA-49 (I79A) and H3A-50 (I79C) had an enhanced, i.e.,superior 4-HBA-producing ability, as compared with HBA-48 (wild type).

Among the transgenic strains for ubiC gene from Cronobacter sakazakii,HBA-52 (I79A) and HBA-53 (X79C) had an enhanced, i.e., superior4-HBA-producing ability, as compared with HBA-51 (wild type). UsingCorynebacterium glutamicum wild strain (as a control having an emptyvector only), a similar experiment was conducted. In this case, 4-HBAproduction was not observed.

The strain highly expressing the V80C mutant of the ubiC gene ofProvidencia rustigianii (HBA-47) showed the highest 4-HBA-production inthe culture supernatant.

As shown above, based on the analysis of ubiC mutants of Pantoeaananatis, Providencia stuartii, Providencia rustigianii, and Cronobactersakazakii, it was revealed that a mutation at V80 (V80A, V8GC) enhanceschorismate-pyruvate lyase activity.

Similarly, in the cases of ubiC genes of Escherichia coli andCronobacter sakazakii, of which the amino acid residue 179 is known tocorrespond to the above V80 based on the results of homology comparison,it was revealed that a similar mutation at the site (I79A, I79C)enhances chorismate-pyruvate lyase activity. Also, it was demonstratedthat the mutation at the site is an important mutation leading to theactivity enhancement of various types of chorismate-pyruvate lyase, andthat a recombinant strain of Corynebacterium glutamicum in which themutant is highly expressed has an enhanced 4-HBA-producing ability.

TABLE 7 Experiment of 4-HBA production from glucose usingCorynebacterium glutamicum transgenic strains for 4-HBA-producing geneAmount of produced Origin of Type of 4-hydroxybenzoic acid4-hydroxybenzoic introduced (mM) Strain Host strain acid-producing genemutation* (After 24 hours) HBA42 Corynebacterium Providencia stuartiiWild type 1.15 HBA43 glutamicum R V80A 1.51 HBA44 V80C 1.83 HBA45Providencia rustigianii Wild type 1.90 HBA46 V80A 2.44 HBA47 V80C 3.03HBA48 Escherichia coli Wild type 0.67 HBA49 I79A 1.42 HBA50 I79C 1.44HBA51 Cronobacter sakazakii Wild type 1.48 HBA52 I79A 2.53 HBA53 I79C2.69 In the column of the “Type of introduced mutation*”, A means “amutation to alanine”, and C means “a mutation to cysteine”. Also, Vmeans “a mutation of valine”, and I means “a mutation of isoleucine”. Inaddition, 80 means “position 80” and 79 means “position 79”.

Corynebacterium glutamicum HBA-47 was deposited in IncorporatedAdministrative Agency National Institute of Technology and Evaluation,NITE Patent Microorganisms Depositary (2-5-8 Kazusakamatari,Kisarazu-shi, Chiba 292-0818 Japan) under Accession Number NITE BP-01849on Apr. 25, 2014. The strain was deposited internationally under theBudapest Treaty and is available to the public under the conditionsspecified in 37 CFR1.808.

INDUSTRIAL APPLICABILITY

According to the present invention, using microorganisms, 4-HBA can beproduced from glucose or the like with a practical efficiency.

1-16. (canceled)
 17. A transformant obtained by introducing, into acoryneform bacterium as a host, a DNA which encodes a mutantchorismate-pyruvate lyase of the following (A) or (B): (A) a mutantchorismate-pyruvate lyase obtained by replacing valine at position 80 ina chorismate-pyruvate lyase (ubiC) from Pantoea ananatis consisting ofthe amino acid sequence of SEQ ID NO: 1 with one or more other aminoacids, (B) a mutant chorismate-pyruvate lyase obtained by replacing anamino acid in another chorismate-pyruvate lyase, the amino acid being ata position enzymologically homologous with that of the above valine,with one or more other amino acids.
 18. The transformant of claim 17,wherein the coryneform bacterium as the host is a Corynebacterium. 19.The transformant of claim 18, wherein the Corynebacterium glutamicum isCorynebacterium glutamicum R (FERM BP-18976), ATCC13032, or ATCC13869.20. A transformant obtained by introducing the following mutant DNA: (C)a mutant DNA obtained by replacing gtc at positions 240 to 242 in achorismate-pyruvate lyase (ubiC) gene of Pantoea ananatis consisting ofthe base sequence of SEQ ID NO: 10 with a DNA segment which encodes oneor more amino acids different from the amino acid encoded by gtc; or (D)a mutant DNA obtained by replacing a DNA segment in a gene which encodesanother chorismate-pyruvate lyase, the DNA segment being at positionscorresponding to the above gtc, with a DNA segment which encodes one ormore amino acids different from the amino acid encoded by the originalDNA segment into a coryneform bacterium as a host.
 21. The transformantof claim 20, wherein the DNA of the above (D) is a mutant DNA obtainedby replacing (iv) gtc at positions 240 to 242 in a chorismate-pyruvatelyase gene of a Providencia bacterium, (v) atc at positions 237 to 239in a chorismate-pyruvate lyase gene of an Escherichia bacterium, or (vi)atc at positions 237 to 239 in a chorismate-pyruvate lyase gene of aCronobacter bacterium, with a DNA segment which encodes one or moreamino acids different from the amino acid encoded by the original DNAsegment.
 22. The transformant of claim 20, wherein the mutant DNA is thefollowing (d), (e), or (f). (d) a mutant DNA obtained by replacing gtcat positions 240 to 242 in a chorismate-pyruvate lyase gene of Pantoeaananatis consisting of the base sequence of SEQ ID NO: 10, achorismate-pyruvate lyase gene of Providencia stuartii consisting of thebase sequence of SEQ ID NO: 11, a chorismate-pyruvate lyase gene ofProvidencia rustigianii consisting of the base sequence of SEQ ID NO:12, a chorismate-pyruvate lyase gene of Providencia sneebia consistingof the base sequence of SEQ ID NO: 13, a chorismate-pyruvate lyase geneof Providencia rettgeri consisting of the base sequence of SEQ ID NO:14, a chorismate-pyruvate lyase gene of Providencia alcalifaciensconsisting of the base sequence of SEQ ID NO: 15, or achorismate-pyruvate lyase gene of Providencia burhodogranarieaconsisting of the base sequence of SEQ ID NO: 16 with a DNA segmentwhich encodes one or more amino acids different from valine, a mutantDNA obtained by replacing atc at positions 237 to 239 in achorismate-pyruvate lyase gene of Escherichia coli consisting of thebase sequence of SEQ ID NO: 17 with a DNA segment which encodes one ormore amino acids different from isoleucine, or a mutant DNA obtained byreplacing atc at positions 237 to 239 in a chorismate-pyruvate lyasegene of Cronobacter sakazakii consisting of the base sequence of SEQ IDNO: 18 with a DNA segment which encodes one or more amino acidsdifferent from isoleucine, (e) a mutant DNA, which consists of a basesequence having the DNA segment introduced by the above replacement andhaving 90% or more of identity with any one of SEQ ID NOs: 10 to 18, andencodes a polypeptide having chorismate-pyruvate lyase activity, (f) amutant DNA which consists of a base sequence having 90% or more ofidentity with any one of SEQ ID NOs: 10 to 18; encodes a polypeptidehaving chorismate-pyruvate lyase activity; and has a replacement of gtcat positions 240 to 242 in any one of SEQ ID NOs: 10 to 16 with a DNAsegment which encodes one or more amino acids different from valine, ora replacement of a DNA segment corresponding to atc at positions 237 to239 in SEQ ID NO: 17 or 18 with a DNA segment which encodes one or moreamino acids different from isoleucine (here, the DNA segment atpositions 240 to 242 in any one of SEQ ID NOs: 10 to 16 corresponding togtc is a DNA which encodes an amino acid at a position enzymologicallyhomologous with that of valine at position 80 of a chorismate-pyruvatelyase encoded by a DNA consisting of the base sequence of any one of SEQID NOs: 10 to 16; and the DNA segment corresponding to gtc at positions237 to 239 in SEQ ID NO: 17 or 18 is a DNA which encodes an amino acidat a position enzymologically homologous with that of isoleucine atposition 79 of a chorismate-pyruvate lyase encoded by a DNA consistingof the base sequence of SEQ ID NO: 17 or 18).
 23. The transformant ofclaim 20, wherein the DNA introduced by the above replacement is gca,tgc, acc, tcc, or aac.
 24. The transformant of claim 20, wherein thecoryneform bacterium as the host is a Corynebacterium.
 25. Thetransformant of claim 25, wherein the Corynebacterium glutamicum isCorynebacterium glutamicum R (FERM BP-18976), ATCC13032, or ATCC13869.26. Corynebacterium glutamicum HBA-47 (Accession Number: NITE BP-01849),which is a transformant of Corynebacterium glutamicum.
 27. A process forproducing 4-hydroxybenzoic acid or a salt thereof, which comprises astep of culturing the transformant of claim 17 in a reaction mixturecontaining at least one starting compound selected from the groupconsisting of a sugar, a compound that can be metabolized into chorismicacid by the transformant, chorismic acid, and a salt thereof, and a stepof recovering 4-hydroxybenzoic acid or a salt thereof from the reactionmixture.
 28. The process of claim 28, wherein the transformant iscultured under aerobic conditions where the transformant does not grow.